Stable composition comprising a PTHrP analogue

ABSTRACT

The present invention provides a storage-stable composition containing a parathyroid hormone-related protein (PTHrP) analogue and methods of using a PTHrP analogue and the PTHrP compositions described herein to treat osteoporosis, to increase bone mass or to increase bone quality. The composition is storage stable, in sterile form, and in general may be stored at room temperature for at least several weeks to allow convenient parenteral administration to human patients.

RELATED APPLICATION

This application is the U.S. National Stage of International ApplicationNo. PCT/US2007/021216, filed Oct. 3, 2007, published in English, andclaims the benefit of U.S. Provisional Application No. 60/848,960, filedon Oct. 3, 2006. The entire teachings of the above application areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Parathyroid hormone-related protein (“PTHrP”) is a 139 to 173 aminoacid-protein. PTHrP and certain analogs are known to be useful toimprove bone mass and quality in the treatment of osteoporosis andrelated disorders. However, the commercial use of these proteins aspharmaceutical agents requires the development of a formulation that isacceptable in terms of storage stability and ease of preparation.

Furthermore, currently available osteoporosis drugs have limitations onsuitable dosage ranges due to the unwanted side-effects, such ashypercalcemia and increased stimulation of bone resorption. Theseunwanted side-effects and resulting dose limitations reduce thebeneficial effects which can be achieved from these drugs. Thus a needexists for compounds which can be administered at a dose which willincrease the beneficial effects without an increase in the unwantedside-effects.

SUMMARY OF THE INVENTION

The present invention provides a storage-stable composition containing aparathyroid hormone-related protein (PTHrP) analogue and methods ofusing those analogues and compositions containing those analogues asdescribed herein to treat osteoporosis, to increase bone mass or toincrease bone quality. The composition is storage stable, in sterileform, and in general may be stored at room temperature for at leastseveral weeks to allow convenient parenteral administration to humanpatients.

In one embodiment, the present invention provides a storage-stablecomposition suitable for administration to a subject (e.g., a human).The composition comprises a PTHrP analogue and an effective amount ofbuffer to maintain the pH of the composition between 2 and 7. In aparticular embodiment, the PTHrP is [Glu^(22,25), Leu^(23,28,31), Aib²⁹,Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2).

In another embodiment, the present invention provides a sealed containercontaining a storage-stable composition suitable for administration to asubject. The composition comprises PTHrP or an analog thereof and aneffective amount of buffer to maintain the pH of the composition between2 and 7. In a particular embodiment, the PTHrP analogue is [Glu^(22,25),Leu^(23,28,31), Aib²⁹, Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2).

In another embodiment, the present invention provides a drug deliverydevice comprising one or more than one single-use container whichcomprises a storage stable composition comprising PTHrP or an analogthereof and an effective amount of buffer to maintain the pH of thecomposition between 2 and 7. In a particular embodiment, the PTHrPanalogue is [Glu^(22,25), Leu^(23,28,31), Aib²⁹,Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.:2).

In another embodiment, the present invention provides a drug deliverydevice comprising one or more than one multi-use container, whichcomprises a storage stable composition comprising PTHrP or an analogthereof and an effective amount of buffer to maintain the pH of thecomposition between 2 and 7. In a particular embodiment, the PTHrPanalogue is [Glu^(22,25), Leu^(23,28,31), Aib²⁹,Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2).

In another embodiment the present invention provides a method oftreating osteoporosis in a subject in need thereof comprisingadministering to the subject a single daily subcutaneous dose of[Glu^(22,25), Leu^(23,28,31), Aib²⁹, Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ IDNO.: 2) in an amount between 40 and 160 μg for a duration of timesufficient to treat the subject, typically between about 3 months to 36months. In some embodiments, the treatment period is between about 3months to 18 months.

In another embodiment the present invention provides a method ofincreasing bone mass or increasing bone quality in a subject in needthereof comprising administering to the subject a single dailysubcutaneous dose of [Glu^(22,25), Leu^(23,28,31), Aib²⁹,Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO. 2) in an amount between 40 and160 μg for a duration of time sufficient to treat the subject, typicallybetween 3 months and 36 months. In some embodiments, the treatmentperiod is between about 3 months to 18 months.

The PTHrP and analogue compositions of the invention exhibit storagestability in terms of hormone composition and activity. Furthermore,these compositions can be administered, in general, in higher dosagesthan currently available osteoporosis drugs, with the reduction orelimination of unwanted side-effects, such as, hypercalcemia orstimulation of bone resorption. This has the advantage of an increase inbeneficial physiological effects due to the increased dosages and canresult in a reduction in the length of treatment time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the stability of SEQ ID NO. 2 over 24 monthsat 5° C. and 25° C. without any chemical stabilizer.

FIG. 2 is a graph showing the stability of lyophilized SEQ ID NO. 2 over24 months at 5° C. 25° C. and 40° C.

DETAILED DESCRIPTION OF THE INVENTION

The sequence of native hPTHrP (1-34) is as follows:

Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp LeuArg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His Thr Ala (SEQ IDNO: 1).

In a particular embodiment, the PTHrP analogue is [Glu^(22,25),Leu^(23,28,31), Aib²⁹, Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2).

Other PTHrP analogues are described in U.S. Pat. Nos. 6,921,750,5,955,574, 6,544,949, 5,723,577, and 5,696,095 the entire contents ofeach of which are incorporated herein by reference.

A “buffer” as used herein is any acid or salt combination which ispharmaceutically acceptable and capable of maintaining the compositionof the present invention within a desired pH range. Buffers in thedisclosed compositions maintain the pH in a range of about 2 to about 7,about 3 to about 6, about 4 to about 6, about 4.5 to about 5.6, or about5.1. Suitable buffers include, any pharmaceutical acceptable buffercapable of maintaining the above pH ranges, such as, for example,acetate, tartrate phosphate or citrate buffers. In one embodiment, thebuffer is an acetate or tartrate buffer. In another embodiment thebuffer is an acetate buffer. In one embodiment the buffer is acetic acidand sodium acetate.

In the disclosed compositions the concentration of buffer is typicallyin the range of about 0.1 mM to about 1000 mM, about 0.2 mM to about 200mM, about 0.5 mM to about 50 mM, about 1 mM to about 10 mM or about 6mM.

As used herein, an anti-microbial agent is a pharmaceutically acceptablepreservative, suitable for administration to a subject, which inhibits,prevents or delays the growth or micro organisms including, for examplebacteria, viruses and fungi in the compositions of the presentinvention. Suitable anti-microbial agents for use in the compositionsand methods of the present invention include, but are not limited to,cresols, benzyl alcohol, phenol, benzalkonium chloride, benzethoniumchloride, chlorobutanol, phenylethyl alcohol, methyl paraben, propylparaben, thiomersal and phenylmercuric nitrate and acetate. In oneembodiment the anti-microbial agents is m-cresol, chlorocresol orphenol. In another embodiment the anti-microbial agents is chlorocresolor phenol. In another embodiment the anti-microbial agents is phenol.

As used herein an effective amount of an anti-microbial agent is anamount effective to inhibits, prevents or delays the growth or microorganisms including, for example bacteria, viruses and fungi in thecompositions of the present invention. In the compositions of thepresent invention, the amount of anti-microbial agent is typically inthe range from about 0.1 to about 20 mg/ml, about 0.2 to about 30 mg/ml,about 0.2 to about 10 mg/ml, about 0.25 to about 5 mg/ml, about 0.5 toabout 50 mg/ml, about 1 to about 10 mg/ml, about 3 mg/ml or about 5mg/ml.

The compositions of the present invention typically are ready toadminister, aqueous solutions which are sterile, storage-stable andpharmaceutically acceptable without the need for reconstitution prior toadministration. The compositions of the present invention are suitablefor administration to a subject which means that they arepharmaceutically acceptable, non-toxic, do not contain any componentswhich would adversely affect the biological or hormonal effects of thepeptide. The compositions of the present invention do not, for example,comprise any cells.

As used herein a composition of the present invention is storage-stableif the amount, purity of the PTHrP remains above about 95% of theoriginal amount under one of the following conditions: (1) storage forover 2 years at 5° C.; or (2) storage for over 30 days at 25° C.

The compositions are typically stored in a sealed container, vial orcartridge which is typically suitable for long term storage. “Suitablefor long-term storage” means that the vial, container or cartridge doesnot allow for the escape of components of the compositions of thepresent invention or the ingress of external components, such as, microorganisms when kept for at least 3 months at 25° C.

The compositions of the present invention are preferably administered byinjection, typically subcutaneous injection.

The compositions of the present invention, can be stored in single-doseor multi-dose sealed containers, vials or cartridges. The sealedcontainer, vial or cartridge is typically suitable for use with a singleor multi-dose injection pen or drug delivery device, which typicallyallows the patient to administer the peptide themselves. The sealedcontainer can comprise one or more doses of the peptide of the presentinvention, wherein each dose comprises an effective amount of thepeptide as described herein.

A single-dose injection pen, or drug delivery device is typically adisposable device which uses a sealed container which comprises a singledose of an effective amount of a PTHrP in the compositions describedherein. A multi-dose injection pen or drug delivery device typicallycontains more than one dose of an effective amount of a PTHrP thereof inthe compositions described herein. The multi-dose pen can typically beadjusted to administer the desired volume of the storage stablecompositions described herein. In certain embodiment the multi-doseinjection pen prevents the ingress of microbial contaminants fromentering the container or cartridge which can occur through multipleuses of one needle.

Injection pens, as used herein, can also comprise two containers one ofwhich contains a PTHrP, as described herein, in a lyophilized powder, asdescribed below, and the second container contains a liquid forreconstitution of the lyophilized powder. The contents of the twocontainers can be mixed prior to administration.

As discussed above the compositions of the present invention can beadministered by injection. Suitable volumes of the compositions of thepresent invention for injection include about 0.5 to about 1 ml, about0.1 to about 1 ml, about 0.02-to about 0.04 ml, about 0.1-to about 5.0μl, or about 0.1-to about 1.0 μl.

In the compositions of the present invention the concentration of thepeptides is from about 20 mg/ml to about 20,000 mg/ml, from about 100mg/ml to about 10,000 mg/ml, from about 300 mg/ml to about 300 mg/ml,from about 500 mg/ml to about 2000 mg/ml and about 2 mg/ml.

The compositions of the present invention can also be lyophilized usinglyophilization techniques known in the art and stored as a powder whichcan be reconstituted prior to administration. The term “lyophilization”as used herein is a freeze drying or dehydration technique whichinvolves removing a solvent, preferably a water miscible solvent, morepreferably water from a composition or the present invention, typicallyby sublimation under high vacuum when the composition is in a frozenstate. Typically, lyophilization is carried out in lyophilizationequipment (a lyophilizer), which comprises a drying chamber withvariable temperature controls, a condenser to collect water, and avacuum system to reduce the pressure in the drying chamber.

The terms “lyophilized composition”, as used herein mean the solidresidue or powder which is produced or which remains after thelyophilization procedure as defined above. The lyophilized compositionof the present invention typically further comprise a pharmaceuticallyacceptable excipient. The term “pharmaceutically acceptable excipient”as used herein refers to a substance which is added to a solution priorto lyophilization to enhance characteristics such as the color, texture,strength, and volume of the lyophilized cake. Pharmaceuticallyacceptable excipients may be, for example, buffers and pH adjusters,crystalline bulking excipients, stabilizers, and tonicity raisingagents.

In certain preferred embodiments the pharmaceutically acceptableexcipient is a crystalline bulking excipient. The terms “crystallinebulking excipient” or “crystalline bulking agent” as used herein meansan excipient which provides bulk and structure to the lyophilizationcake. These crystalline bulking agents are inert and do not react withthe peptide. In addition, the crystalline bulking agents are capable ofcrystallizing under lyophilization conditions.

Examples of suitable crystalline bulking agents include hydrophilicexcipients, such as, water soluble polymers; sugars, such as mannitol,sorbitol, xylitol, glucitol, ducitol, inositiol, arabinitol, arabitol,galactitol, iditol, allitol, maltitol, fructose, sorbose, glucose,xylose, trehalose, allose, dextrose, altrose, lactose, glucose,fructose, gulose, idose, galactose, talose, ribose, arabinose, xylose,lyxose, sucrose, maltose, lactose, lactulose, fucose, rhamnose,melezitose, maltotriose, raffinose, altritol, their optically activeforms (D-or L-forms) as well as the corresponding racemates; inorganicsalts, both mineral and mineral organic, such as, calcium salts, such asthe lactate, gluconate, glycerylphosphate, citrate, phosphate monobasicand dibasic, succinate, sulfate and tartrate, as well as the same saltsof aluminum and magnesium; carbohydrates, such as, the conventionalmono-and di-saccharides as well as the corresponding polyhydricalcohols; proteins, such as, albumin; amino acids, such as glycine;emulsifiable fats and polyvinylpyrrolidone. Preferred crystallinebulking agents are selected from the group consisting of glycine,mannitol, dextran, dextrose, lactose, sucrose, polyvinylpyrrolidone,trehalose, glucose and combinations thereof. Particularly useful bulkingagents include dextran.

As used herein a stabilizer is a composition which maintains thechemical, biological or hormonal stability of the peptide. Examples ofstabilizing agent include polyols which includes a saccharide,preferably a monosaccharide or disaccharide, e.g., glucose, trehalose,raffinose, or sucrose; a sugar alcohol such as, for example, mannitol,sorbitol or inositol, a polyhydric alcohol such as glycerine orpropylene glycol or mixtures thereof and albumin.

The compositions described herein can be used to stimulate bone growthin a subject. Thus they are useful in the treatment of diseases ordisorders associated with deficiency in bone growth such as osteoporosisand bone fractures. In one embodiment, the present invention is a methodof treating osteoporosis in a subject comprising administering to thesubject an effective amount of composition described herein.

As used herein, “treating” can include both prophylactic, andtherapeutic treatment. For example, therapeutic treatment can includedelaying inhibiting or preventing the progression of osteoporosis, thereduction or elimination of symptoms associated with osteoporosis.Prophylactic treatment can include preventing, inhibiting or delayingthe onset of osteoporosis.

As used herein, an effective amount refers to an amount sufficient toelicit the desired response. In the present invention, the desiredbiological response is an decrease in the rate of bone loss and/or anincrease in the bone mass or bone quality of a subject.

Suitable dosage for use in the compositions and methods of the presentinvention include from about 40 to about 160 μg, about 80 to about 120μg about 80 to about 100 μg; or from about 40 to about 50 μg, about 50to about 60 μg, about 60 to about 70 μg, about 70 to about 80 μg, about80 to about 90 μg, about 90 to about 100 μg, about 100 to about 110 μg,about 110 to about 120 μg, about 120 to about 130 μg, about 130 to about140 μg, about 140 to about 150 μg, about 150 to about 160 μg; or from 40to about 45 μg, about 45 to about 50 μg, about 50 to about 55 μg, about55 to about 60 μg, about 60 to about 65 μg, about 65 to about 70 μg,about 70 to about 75 μg, about 75 to about 80 μg, about 80 to about 85μg, about 85 to about 90 μg, about 90 to about 95 μg, about 95 to about100 μg, about 100 to about 105 μg, about 105 to about 110 μg, about 110to about 115 μg, about 115 to about 120 μg, about 120 to about 125 μg,about 125 to about 130 μg, about 130 to about 135 μg, about 135 to about140 μg, about 140 to about 145 μg, about 145 to about 150 μg, about 150to about 155 μg, about 155 to about 160 μg administered once per day,once every other day, twice per week once per week, once every twoweeks, once per month. The doses can be a pulsatile injection, forexample, once per month which causes pulsatile release of singles dosesof the composition described herein.

When the dosages described above are administered once per day, once perweek etc., typically the dosages are of equal amounts.

The subject as used herein can be an animal, for example, a mammal, suchas a human.

A pharmaceutically acceptable salt is a salt which is suitable foradministration to a subject, such as, a human. The peptides of thepresent invention can have one or more sufficiently acidic proton thatcan react with a suitable organic or inorganic base to form a baseaddition salt. Base addition salts include those derived from inorganicbases, such as ammonium or alkali or alkaline earth metal hydroxides,carbonates, bicarbonates, and the like, and organic bases such asalkoxides, alkyl amides, alkyl and aryl amines, and the like. Such basesuseful in preparing the salts of this invention thus include sodiumhydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate,and the like. The peptides of the present invention having asufficiently basic group, such as an amine can react with an organic orinorganic acid to form an acid addition salt. Acids commonly employed toform acid addition salts from compounds with basic groups are inorganicacids such as hydrochloric acid, hydrobromic acid, hydroiodic acid,sulfuric acid, phosphoric acid, and the like, and organic acids such asp-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, acetic acid, and the like. Examples of such salts includethe sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,decanoate, caprylate, acrylate, formate, isobutyrate, caproate,heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate,hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate,phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate,gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate,propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,mandelate, and the like.

The compositions of the present invention typically do not show any orshow reduced side-effects such as hypercalcemia and typically do notincrease the stimulation of bone resorption at the dosage listed above.This reduction in side effects allows for administration of higher dosesthan commercially available osteoporosis drugs.

The compositions of the present invention can be administered byinjection as described herein.

The compositions of the present invention may be administered alone orin combination with an additional therapeutic agent, such as anantiresorptive therapy, for example, bisphonsphonates and calcitonin.

EXEMPLIFICATION Example 1 Demonstrates [Glu^(22,25), Leu^(23,28,31),Aib²⁹, Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2) Stability at LowAcetate Concentration (1 mM), Without Stabilizer

TABLE 1 Unitary Formula Material Supplier (per cartridge) (SEQ ID NO.:2) Ipsen Ireland 0.140 mg (free base) Tri-hydrate sodium acetate 0.1 NProlabo 14.6 mg Acetic acid 0.1 N Prolabo 1.9 mg qs pH 5.1 Water forInjection Meram qs 1.4 g Type I clear glass Cartridge Bünderglass via 11.5 ml, washed, siliconised Vetter and sterilised Grey PTFE bromobutylcartridge Daïkyo 1 rubber stopper Chlorobutyl rubber-metal West 1cartridge crimp Pharmaceutical qs = quantity sufficient to achieve

The formulation delivered 100 mcg of (SEQ ID NO.: 2) per 0.1 ml. (SEQ IDNO.: 2) was dissolved in Water for Injection containing dilute acetatebuffer to give pH 5.1 was used.

Results confirm excellent chemical stability over 24 months, at 5° C. asshown in FIG. 1. This solution contains no stabilizer or preservativeand only 6 mM acetate buffer.

In summation for (SEQ ID NO.: 2), stabilizer is not needed to give goodstability in solution.

Example 2 Use of Citric Acid Buffer in Lyophilized Form of (SEQ. ID NO.:2)

TABLE 2 Unitary Formula Material Supplier (per vial) (SEQ ID NO.: 2)Ipsen Ireland 0.1 mg (free base) Dextran 70 Interchemical 50 mg Citricacid 0.25% (w/v) Prolabo qs pH 4.5* Water for injections** Meram qs 1 gType I clear glass vial, 11-13 ml Verretubex 1 Grey chlorobutyl PTFEstopper, Daïkyo 1 20 mm Flip-off metal crimp West Pharma 1 **to get pH5-5.5 after lyophilisation removed after freeze-drying step.

The solutions in TABLE 2 were reconstituted with NaCl 0.9%, to give:

ONE vial of 2 ml (=50 μg/ml) providing 10 to 80 μg/d doses (withinjections of 200 μl to 1.6 ml), or

ONE vial of 5 ml (=20 μg/ml solution) providing 5 to 40 μg/d doses (withinjections of 250 μl-2 ml).

Citric acid was used to adjust pH and Dextran was used to provide abulking agent to aid cake formation during lyophilization.

The solutions described were lyophilized in glass vials, and stored atvarious temperatures for up to 24 months. The content of [Glu^(22,25),Leu^(23,28,31), Aib²⁹, Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2),purity and physical tests were conducted on samples removed from storageat different times. Results are presented in FIG. 2, for peptideconcentration, as percent remaining. The data in FIG. 2 shows excellentstability over 24 months at 2-8° C.

Example 3 Screening of Formulations for [Glu^(22,25), Leu^(23,28,31),Aib²⁹, Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2) to Compare DifferentPreservatives

TABLE 3 below shows Methyparaben and Benzyl Alcohol are not suitablepreservatives for use with [Glu^(22,25), Leu^(23,28,31), Aib²⁹,Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2), as precipitation and/orinactivity in preservative activity was seen.

TABLE 3 Example 3a Example 3b Example 3c Example 3d Example 3eMethylparaben 1.5 mg/mL 1.35 mg/mL — — — Propylparaben — 0.15 mg/mL — —— Phenol — — 5 mg/mL — — Chlorocresol — — — 3 mg/mL — Benzyl alcohol — —— — 10 mg/mL Preservative Failed Pass Pass Pass Pass effectiveness testObservationo or Precipitation — — — — Issues observed Preservative NotTested Pass Pass Pass Fail effectiveness as test after storageprecipitated 4.5 months at 5° C. initially

Solutions were prepared containing [Glu^(22,25), Leu^(23,28,31), Aib29,Lys^(26,31)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2) 2 mg/ml, acetate buffer 6 mMand water for injection, with various different preservatives added atconcentrations recommended for effective antimicrobial activity.Solutions were prepared at room temperature, by dissolution of thevarious ingredients in water for injection, with stirring over <30minutes to ensure complete dissolution, Solutions were filtered through0.2 micron filter and filled into glass vials, to which a rubber stopperwas applied and crimped in place to ensure complete closure.

The solution with methylparaben was less acceptable due to precipitationand inactivity immediately after manufacture of the solution. Thesolutions were then stored for up to 3 months at 25° C., and up to 4.5months at 5° C. and the preservative effectiveness test repeated. asdescribed in Example 5.

Example 4 Evaluation of Anti-Microbial Preservative Effectiveness ofVarious Concentrations of [Glu^(22,25), Leu^(23,28,31), Aib²⁹,Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2) Compositions (StabilityStudy)

TABLE 4 P87228 P87229 P87230 P87231 (SEQ ID 2 mg/mL 2 mg/mL 2 mg/mL  2mg/mL NO.: 2) Anti- Phenol Chlorocresol Chlorocresol Benzyl alcoholmicrobial 5 mg/mL 3 mg/mL 2 mg/mL 10 mg/mL Acetate pH 5.1 pH 5.1 pH 5.1pH 5.1 buffer

The solutions were tested according to European Pharmacopoeia, Chapter5.1.3 “Efficacité de la conservation anti-microbienne” (Anti-microbialeffectiveness test) to prove the effectiveness of the preservative.

TABLE 5 Preservative effectiveness test after manufacturing Nb of cfupresent/preparation Initial (plate-count method) organism P87228 P87229P87230 P87231 Organisms: concentration Test Phenol ChlorocresolChlorocresol Benzyl alc. Bacteria in cfu/mL interval 5 mg/ml 3 mg/ml 2mg/ml 10 mg/ml Staphylococcus 3.8 × 10⁵ T 0 3.4 × 10⁵ <5 <5 4.7 × 10⁵aureus T + 6 hrs <5 <5 <5 6.8 × 10² T + 24 hrs <5 <5 <5 <5 T + 28 days    5 (*) <5 <5 <5 Pseudomonas 1.3 × 10⁶ T 0  5 <5 <5 1.5 × 10²aeruginosa T + 6 hrs <5 <5 <5 <5 T + 24 hrs <5 <5 <5 <5 T + 28 days <5<5 <5 <5 E. coli 6.7 × 10⁵ T 0 7.2 × 10³ <5 <5 1.1 × 10⁵ T + 6 hrs <5 <5<5 <5 T + 24 hrs <5 <5 <5 <5 T + 28 days <5 <5 <5 <5 Nb of cfupresent/preparation Initial (plate-count method) organism P87228 P87229P87230 P87231 Organism: concentration Test Phenol chlorocresolchlorocresol Benzyl alc. Yeast and mold in cfu/mL interval 5 mg/ml 3mg/ml 2 mg/ml 10 mg/ml Aspergillus 3.4 × 10⁵ T 0 4.0 × 10⁵ <5 <5 4.1 ×10⁵ niger T + 7 days <5 <5 <5 <5 T + 28 days <5 <5 <5 <5 Candida 3.9 ×10⁵ T 0 4.4 × 10⁵ <5 <5 3.8 × 10⁵ albicans T + 7 days <5 <5 <5   5 T +28 days <5 <5 <5 <5 Results: Conform — Conform Conform Conform ConformPreservative effectiveness test results after 3 months storage at 25° C.Nb of cfu present/preparation Initial (plate-count method) organism TestP87228 P87229 P87230 P87231 Organisms: concentration interval Phenolchlorocresol chlorocresol Benzyl alc. Bacteria in cfu/mL (days) 5 mg/ml3 mg/ml 2 mg/ml 10 mg/ml Staphylococcus 2.7 × 10⁵ 0 hr 1.9 × 10⁵ <5 <53.8 × 10⁵ aureus (P87228, 6 hr 30 <5 <5 5.9 × 10³ P87229, P87231) 24 hr<5 <5 <5 <5 5.2 × 10⁵ 28 day <5 <5 <5 <5 (P87230) Pseudomonas 9.9 × 10⁵0 hr <5 <5 <5 <5 aeruginosa (P87228, 6 hr <5 <5 <5 <5 P87229, P87231) 24hr <5 <5 <5 <5 8.5 × 10⁵ 28 day <5 <5 <5 <5 (P87230) E. coli 6.8 × 10⁵ 0hr 1.7 × 10⁵ <5 <5 8.0 × 10⁴ (P87228, 6 hr <5 <5 <5  5 P87229, P87231)24 hr <5 <5 <5 <5 9.5 × 10⁵ 28 day <5 <5 <5 <5 (P87230) Nb of cfupresent/preparation Initial (plate-count method) organism P87228 P87229P87230 P87231 Organism: concentration Test Phenol ChlorocresolChlorocresol Benzyl alc. Yeast and mold in cfu/mL interval 5 mg/ml 3mg/ml 2 mg/ml 10 mg/ml Aspergillus 3.3 × 10⁵ 0 hr 3.8 × 10⁵ 55 70 4.1 ×10⁵ niger (P87228, 7 day <5 <5 <5 <5 P87229, P87231) 28 day <5 <5 <5 <54.1 × 10⁵ (P87230) Candida 2.7 × 10⁵ 0 hr 4.0 × 10⁵ <5 <5 3.8 × 10⁵albicans (P87228, 7 day <5 <5 <5 <5 P87229, P87231) 28 day <5 <5 <5 <53.7 × 10⁵ (P87230) Results: Conform — Conform Conform Conform NotConform Preservative effectiveness test results after 4.5 months storageat 5° C. Nb of cfu present/preparation Initial (plate-count method)organism Test P87228 P87229 P87230 P87231 Organisms: concentrationinterval Phenol chlorocresol chlorocresol Benzyl alc. Bacteria in cfu/mL(days) 5 mg/ml 3 mg/ml 2 mg/ml 10 mg/ml Staphylococcus 5.4 × 10⁵ 0 hr4.1 × 10⁵ <5 <5 5.1 × 10⁵ aureus 6 hr <5 <5 <5 7.2 × 10³ 24 hr <5 <5 <5<5 28 day <5 <5 <5 <5 Pseudomonas 9.7 × 10⁵ 0 hr <5 <5 <5 <5 aeruginosa6 hr <5 <5 <5 <5 24 hr <5 <5 <5 <5 28 day <5 <5 <5 <5 E. coli 6.1 × 10⁵0 hr 7.0 × 10⁴  5  5 4.2 × 10⁴ 6 hr <5 <5 <5 <5 24 hr <5 <5 <5 <5 28 day<5 <5 <5 <5 Nb of cfu present/preparation Initial (plate-count method)organism P87228 P87229 P87230 P87231 Organism: concentration Test PhenolChlorocresol Chlorocresol Benzyl alc. Yeast and mold in cfu/mL interval5 mg/ml 3 mg/ml 2 mg/ml 10 mg/ml Aspergillus 5.3 × 10⁵ 0 hr 3.7 × 10⁵1.8 × 10³ 7.5 × 10³ 4.1 × 10⁵ niger 7 day <5 <5 <5 <5 28 day <5 <5 <5 <5Candida 4.1 × 10⁵ 0 hr 4.5 × 10⁵ <5  5 4.5 × 10⁵ albicans 7 day <5 <5 <5<5 28 day <5 <5 <5 <5 Results: Conform — Conform Conform Conform NotConform (*) Bacillus Gram +, different from St. Aureus −> result conformNb of cfu = number of colony forming units

TABLE 5 shows Phenol, Chlorocresol and Benzyl Alcohol all producecompliant results immediately after manufacture for both Bacteria andYeasts/moulds. After 3 and 4.5 months storage, the preservative efficacyis maintained for Phenol and Chlorocresol, for both Bacteria andYeasts/moulds. However, for Benzyl Alcohol, the efficacy againstBacteria is not compliant, as the data shows insufficient rate of killagainst S. Aureus (TABLE 5).

Example 5 Chemical Stability of Different Formulations

TABLE 6 details the chemical stability of the formulations described inExample 4.

TABLE 6 Glu^(22, 25), Leu^(23, 28, 31), Aib²⁹,Lys^(26, 30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2) stability results Storageconditions: 25° C., 60% RH (SEQ ID NO.: 2) content in mg/mL (% initialconcentration at t = 0) Batch Composition 0 month 1 month 3 monthsP87228 (SEQ ID NO.: 2) 1.90 1.88 1.83 (2 mg/ml)/Phenol (100%) (98.9%)(96.3%) (5 mg/ml) P87229 (SEQ ID NO.: 2) 1.98 1.96 1.94 (2mg/ml)/Chlorocresol (100%) (99.0%) (98.0%) (3 mg/ml) P87231 (SEQ ID NO.:2) 1.93 1.89 1.86 (2 mg/ml)/Benzyl (100%) (97.9%) (96.4%) Alcohol (10mg/ml) Storage conditions: 5° C. (SEQ ID NO.: 2) content in mg/mL (%initial concentration at t = 0) Batch Composition 0 month 3 month 4.5month P87228 (SEQ ID NO.: 2) 1.90 1.91 1.89 (2 mg/ml)/Phenol (100%)(100.5%) (99.5%) (5 mg/ml) P87229 (SEQ ID NO.: 2) 1.98 1.96 1.97 (2mg/ml)/Chlorocresol (100%) (99.0%) (99.5%) (3 mg/ml) P87231 (SEQ ID NO.:2) 1.93 1.94 1.92 (2 mg/ml)/Benzyl (100%) (100.5%) (99.5%) Alcohol (10mg/ml)

As can be seen from TABLE 6 and [Glu^(22,25), Leu^(23,28,31), Aib²⁹,Lys^(26,30)]hPTHrP(1-34)NH₂ (SEQ ID NO.: 2) solution stability is notsignificantly influenced by the preservative selected. TABLE 7 detailsthe content of each preservative for the same formulations.

TABLE 7 Preservative stability results Storage conditions: 25° C., 60%RH Preservative content in mg/ml (% initial concentration at t = 0)Batch Composition 0 month 1 month 3 month P87228 (SEQ ID NO.: 2) 4.864.82 4.79 (2 mg/ml)/Phenol (100%) (99.2%) (98.6%) (5 mg/ml) P87229 (SEQID NO.: 2) 2.78 2.70 2.56 (2 mg/ml)/Chlorocresol (100%) (97.1%) (92.1%)(3 mg/ml) P87231 (SEQ ID NO.: 2) 9.92 9.83 9.82 (2 mg/ml)/Benzyl (100%)(99.1%) (99.0%) Alcohol (10 mg/ml) Storage conditions: 5° C.Preservative content in mg/mL (% initial concentration at t = 0) BatchComposition 0 month 3 month 4.5 month P87228 (SEQ ID NO.: 2) 4.86 4.834.84 (2 mg/ml)/Phenol (100%) (99.4%) (99.6%) (5 mg/ml) P87229 (SEQ IDNO.: 2) 2.78 2.73 2.74 (2 mg/ml)/Chlorocresol (100%) (98.2%) (98.6%) (3mg/ml) P87231 (SEQ ID NO.: 2) 9.92 9.89 9.94 (2 mg/ml)/Benzyl (100%)(99.7%) (100.2%) Alcohol (10 mg/ml)

As can be seen from TABLE 7 chlorocresol is the preservative which hasthe lower stability, with greater loss in preservative content underboth 5 and 25° C. storage.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A storage-stable composition suitable foradministration to a subject comprising: a) a PTHrP analogue having thesequence [Glu^(22,25), Leu^(23,28,31), Aib²⁹, Lys^(26,30)]hPTHrP(1-34)NH₂(SEQ ID NO.2); and b) an effective amount of a pH bufferto maintain the pH in a range of about 4.5 to about 5.6.
 2. Thestorage-stable composition according to claim 1, wherein said pH isabout 5.1.
 3. The storage stable composition according to claim 1,wherein said pH buffer is selected from the group consisting of acetate,tartrate, phosphate and citrate buffers.
 4. The storage-stablecomposition according to claim 3, wherein said pH buffer is an acetatebuffer.
 5. The storage-stable composition according to claim 4, whereinsaid acetate buffer is acetic acid and sodium acetate.
 6. Thestorage-stable composition according to claim 5, wherein said buffer ispresent in a concentration range of about 1 mM to about 10 mM.
 7. Thestorage stable composition according to claim 6 wherein said buffer ispresent in a concentration of about 6 mM.
 8. The storage-stablecomposition according to claim 1, further comprising an effective amountof an anti-microbial agent.
 9. The storage-stable composition accordingto claim 8, wherein said anti-microbial agent is phenol.
 10. Thestorage-stable composition according to claim 9, wherein said phenol ispresent in a concentration from about 0.25 to about 5 mg/mL.
 11. Thestorage-stable composition according to claim 10, wherein said phenol ispresent in a concentration of about 5 mg/mL.
 12. The storage-stablecomposition according to claim 1, wherein said PTHrP analogue is presentin a concentration of about 2 mg/mL.
 13. The storage-stable compositionaccording to claim 1, wherein said composition does not contain achemical stabilizer.